Enzymatic conversion of dehydrocoelenterazine to coelenterazine using FMN-bound flavin reductase of NAD(P)H:FMN oxidoreductase
Enzymatic conversion of dehydrocoelenterazine to coelenterazine using FMN-bound flavin reductase of NAD(P)H:FMN oxidoreductase
复制标题
使用 NAD(P)H:FMN 氧化还原酶的 FMN 结合黄素还原酶将脱氢腔肠素酶促转化为腔肠素
DOI：
10.1016/j.bbrc.2021.11.089
复制
发表时间：
2022
影响因子：
3.1
通讯作者：
Hosoya Takamitsu
中科院分区：
文献类型：
--
作者：
Inouye Satoshi;Nakamura Mitsuhiro;Hosoya Takamitsu
腔肠素（CTZ）是海洋生物中与荧光素酶（一种酶）发生发光反应的底物，在水溶液中不稳定。脱氢形式的CTZ（脱氢腔肠素，dCTZ）是稳定的，并且被认为是CTZ的储存形式和从腔肠素和4-羟基苯基戊酸缩合反应到CTZ的再循环中间体。在本研究中，在NADH存在下，使用来自生物发光细菌Vibrio fischeriATCC 7744（FRase）的NAD（P）H：FMN氧化还原酶成功地实现了dCTZ到CTZ的酶促转化（FRase-NADH反应）。通过HPLC和LC/ESI-TOF-MS分析鉴定FRase-NADH反应中从dCTZ还原的CTZ。因此，dCTZ可以在体外被酶促转化为CTZ，并且dCTZ的浓度可以通过利用CTZ的荧光酶（Gaussialuciferase或Renillaluciferase）与FRase-NADH反应偶联的发光活性来确定。
Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacteriumVibrio fischeriATCC 7744 (FRase) in the presence of NADH (the FRase–NADH reaction). CTZ reduced from dCTZ in the FRase–NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZin vitro.Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussialuciferase orRenillaluciferase) coupled with the FRase–NADH reaction.